Transgenic, non-human animals can be used to understand the action of a single gene or genes in the context of the whole animal and the interrelated phenomena of gene activation, expression, and interaction. The technology has also led to the production of models for various diseases in humans and other animals which contributes significantly to an increased understanding of genetic mechanisms and of genes associated with specific diseases.
Traditionally, smaller animals such as mice have been used as disease models for human diseases and have been found to be suitable as models for certain diseases. However, their value as animal models for many human diseases is quite limited due to differences in mice compared to humans. Larger transgenic animals are much more suitable than mice for the study of many of the effects and treatments of most human diseases because of their greater similarity to humans in many aspects. Particularly, pigs are believed to be valuable as disease models for human diseases.
Integration of foreign DNA plays a pivotal role in both genetic manipulation of cell lines and technologies related to therapeutic gene transfer. Current integrations strategies, based upon for example retroviral, lentiviral or DNA transposon-based vector systems allow efficient gene insertion, but all suffer from the fact that gene insertion is not controllable and cannot be directed to predetermined positions in the genomic DNA. The yeast Flp recombinase, in contrast, facilitates sequence-specific integration (1), but the Flp recombination target sequence (FRT) does not exist in mammalian genomes. The site of integration is of great importance for the gene expression profile of the inserted gene. Hence, in some positions the gene will be stably expressed, whereas other positions are unable to support long-term expression due to strong influences from the flanking DNA leading to transcriptional silencing. Such actions upon the transgene may lead to reduced expression or complete shut-down of expression depending on cell type or tissue. For several purposes it is therefore of great importance to direct insertion towards ‘stably’ expressing loci. This may have particular importance in genetically manipulated animal models in which continued gene expression in the tissue of interest is essential for genetic studies. As another important example, cell therapies in which genetically altered effector cells are administered to patients (as in some cancer immunotherapy protocols) rely on stable transgene expression from loci that are not silenced over time.
The tyrosine recombinases Flp (2) and Cre, derived from yeast and E. coli phages, respectively, and the serine recombinase φC31 from S. lividans phages are cherished for their site-specific integrating properties. φC31 has been found to facilitate plasmid DNA recombination into pseudo recognition sites in the human genome and therefore has been extensively explored as a tool in gene therapy (3). In case of Flp and Cre, however, the human genome does not contain recombination target sites and these sites need to be introduced in the genome prior to successful gene insertion (1). Although Cre-based recombination has been heavily studied and appears to be a bit more effectful than Flp in human cells, a now widely used Flp-based integration system has been commercialized by Invitrogen (cat. no. K6010-01). This system is based on a FRT sequence contained within a lacZ-Zeocin fusion gene. This FRT-tagged gene is inserted into cells by nonhomologous recombination, an uncontrolled recombination process which is believed often to involve concatamer formation, leading to insertion of more than one copies of the foreign DNA. Characterized cell lines containing this FRT-lacZzeo insert are currently offered by Invitrogen, allowing researchers to insert plasmid DNA containing their gene of interest into the FRT-tagged locus on offer in the particular cell line. This plasmid contains not only the transgene but also a FRT-hygro cassette that does not contain a start codon. By recombination between the two FRT sites (one in the genome and one on the plasmid) the start codon of the lacZzeo fusion is fused to the FRT-hygro cassette, allowing for expression of the hygro gene and subsequent selection for hygromycin B resistance. This technology facilitates insertion of the entire plasmid including the bacterial backbone which is believed to have a negative impact on gene expression in mammalian cells potentially be inducing posttranscriptional silencing.
Transcriptional silencing of foreign genetic material is a fundamental problem in gene transfer and genetic engineering of cells and animals. Due to epigenetic modifications transgenic animal models therefore often suffer from reduced gene expression, or the lack of gene activity in tissues in which transcription is required to develop a desired phenotype. The choice of promoter influences the overall transgene expression profile in a transgenic animal and to a certain degree the level of gene silencing. However, positional effects and spreading of heterochromatin from flanking genomic regions are major contributors to gene silencing, and the site of integration of a transgene is crucial, therefore, for the fate of a foreign gene. In rodents, well-characterized loci supporting long-term gene expression have been identified. Based on these findings transgenic animal models have been generated by inserting genes by homologous recombination into such preferred sites.
The establishment of cloned pig models of genetic disease, is challenged by problems in identifying genomic loci that support ubiquitous or, for some models, tissue-specific expression of an inserted transgene. At present, the information that allows the insertion of genes into well-suited and predefined loci of porcine cells is not available. Moreover, by inserting disease genes at random positions we risk to target genomic sites that are eventually silenced during pig development and growth. Therefore, a need exists for a genetically modified pig harbouring an insertion site that allows for the integration of a transgene at a position in the genome wherein the transgene is stably expressed.